Artificial invaplex

ABSTRACT

An artificial invasin complex is prepared from purified or recombinantly prepared invasins and gram negative bacteria lipopolysaccharides. Typically, IpaB is mixed with IpaC to form a IpaB:IpaC complex. This invasin protein complex is then mixed with the lipopolysaccharide to form an artificial invasin complex. Additional bioactive molecules can be incorporated into the complex during manufacture. This artificial invasin complex is similar in function to native Invaplex 24 or Invaplex 50. The artificial invasin complex has superior immunogenicity properties relative to the native complex and can be tailor made. Its method of preparation lends itself to scale up. The artificial invasin complex can facilitate transport of biomolecules, therapeutics and antibiotics across cell membranes in a manner similar to native  Shigella Invaplex.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention provides a novel method for preparing an artificialInvaplex, an artificial invasin complex comprising at least two invasinproteins and a lipopolysaccharide from invasive gram negative bacteria.The artificial invasin complex of the present invention can be used as avaccine, an adjuvant for vaccines, biochemical, or other substances, andas a diagnostic tool.

2. Description of Related Art

Shigellosis is a leading cause of human diarrheal disease particularlyin developing countries where it is estimated that over 163 millioncases, with 1 million fatal cases, occur annually [1]. The most commonShigella species causing disease worldwide are S. flexneri and S. sonnei[1]. A lower incidence (1.5 million cases/yr) of shigellosis inindustrialized countries [1] implies that the adult population isnon-immune and susceptible. Even in areas relatively free ofshigellosis, massive destruction due to war or natural disasters, suchas the earthquake of 1999 in Kocaeli, Turkey, can suddenly give rise tomultispecies, multi-focal increases in dysentery [2]. Similarly, whendeployed troops, relief workers, or travelers from industrializedcountries are in areas endemic for Shigella or in areas where the localinfrastructure has collapsed, the threat of exposure to multipleShigella spp. may be significant. S. dysenteriae 1 causes the mostsevere form of shigellosis and is often associated with hemolytic uremicsyndrome [3]. Although the incidence of S. dysenteriae 1 is not as highas that of S. flexneri or S. sonnei, epidemics caused by S. dysenteriae1 often occurs every 10 to 12 years [4]. The periodic occurrence isprobably due to many factors, one of which is the likely waning ofimmunity to S. dysenteriae 1. More important is the recent finding thatantibiotic resistance to the flouroquinoline class of antibiotics hasbeen demonstrated in S. dysenteriae 1 isolated in recent outbreaks [5,6]. The likelihood of another epidemic of S. dysenteriae 1 is high andif it is caused by a strain with broad antibiotic resistance includingthe fluorquinolones, the consequences could be severe.

Shigella species are one of the few proven agents for biologicalterrorism. The extremely low infectious dose (for S. dysenteriae 1 it isreported to be 10 bacteria) [7] and the ease of growing the organism insimple bacteriological medium are suitable factors for consideringShigella spp as potent biological weapons. The proven effectiveness as abiological terrorism agent is a case in which employees in a hospitalwere deliberately infected with S. dysenteriae 2 by a disgruntledcoworker who had “painted” muffins with the organism [8]. Otherfood-borne outbreaks of Shigella infections are well documented—fromcold food on airlines to salads on cruise ships [9, 10] demonstratingthe ease by which shigellae can cause disease in a susceptiblepopulation. Prevention of Shigella infections is best accomplished byenvironmental controls to include good sanitation and clean watersupplies. Such infrastructure improvements are easily overcome ifshigellae are deliberately planted in food or drinks. Furthermore, theease of isolating shigellae from nature [5] and the simplicity ofdeveloping antibiotic resistant S. dysenteriae 1 strains in thelaboratory validates this agent as a potential major bioterrorismthreat. Although an initial outbreak might be difficult to completelystop in a non-immune population, it is important to limit secondaryspread. An effective means of prevention are vaccines which areeffective against shigellae regardless of antibiotic resistance.

The pathogenesis of Shigella spp. is attributed to the organism'sability to invade, replicate intracellularly, and spread intercellularlywithin the colonic epithelium. The initial entry of shigellae occursthrough specialized intestinal epithelial cells called M cells. Severalhighly conserved, virulence plasmid-encoded proteins, called theinvasion plasmid antigens (IpaA, IpaB, IpaC, and IpaD), are essentialparticipants in the invasion process and are likely key to theefficiency of Shigella infections. Upon contact or attachment to hostcells, the Shigella invasins are released [11] by a type III secretionapparatus [12] and induce a phagocytic event resulting in engulfment andinternalization of the bacterium by the host cell [13]. The activecomponents include an IpaB:IpaC complex that integrates into the hostcell membrane, forming a channel by which other Shigella proteins gainentry into the host cell [14]. Recently, we have isolated from intact,virulent Shigella cells, an invasin protein-LPS complex which serves asan effective vaccine and adjuvant [15-19]. The Shigella invasin complex(Invaplex) is prepared by extracting intact virulent shigellae withwater followed by anion-exchange chromatography of the water extract.Two fractions, called Invaplex 24 and Invaplex 50, contain the majorantigens including LPS, IpaB and IpaC. The invasin complex (Invaplex)binds to surfaces of cultured epithelial cells whereas similarpreparations from non-invasive shigellae do not exhibit this property.Shortly after attachment the Invaplex becomes localized inside the hostcell via an acting-dependent endocytic event likely induced by theInvaplex. It appears that IpaC and IpaB have a significant role in theattachment and internalization process in that antibodies against IpaCor IpaB inhibit the binding of Invaplex to host cell membranes [20].Antibodies to LPS do not alter the uptake event. Once internalized theInvaplex traffics through early endosomes, late endosomes, then theGolgi apparatus and eventually ends up free in the host-cell cytoplasm.The ability to bind to a eukaryotic host cell surface and stimulateendocytosis indicates that the Invaplex maintains an active, nativevirulence structure similar to that found in invasive Shigella.

Historically, successful Shigella vaccines have emphasized presentationof LPS in an effective manner to elicit protection. Many approaches havebeen used for Shigella vaccines including live attenuated vaccines [21,22], killed whole cell vaccines [23, 24], and delivery of Shigella LPSor O-polysaccharides with carriers such as proteosomes [25], tetanustoxoid [26], ribosomes [27] or Invaplex (17, 18; see below). Only thelive attenuated vaccines utilize the native invasiveness of theshigellae to deliver the LPS and protein antigens to the mucosal immunesystem presumably via the follicle-associated epithelium [14]. Theresidual pathogenicity of the attenuated vaccine strains may limit thisapproach unless further attenuation is achieved [28].

The ability to isolate a putative native surface structure such asInvaplex, which exhibits activities and immunogenicity similar toinvasive shigellae, has significant implications in vaccine design anddevelopment. First, the isolated native complex may enhance delivery tothe appropriate portal of entry (M-cells), similar to that targeted bylive-attenuated vaccine strains. The attachment of Invaplex to hostcells (and likely intestinal M cells or M-like cells in other mucosaltissues) allows the use of relatively low doses of this subcellularcomplex for immunization due to its delivery efficiency. Similar tolive, attenuated vaccines Invaplex contains all major Shigella antigens(including IpaB, IpaC and LPS) and has the potential to stimulate animmune response equivalent to that produced during natural infection,including recognition of epitopes found only in native structures. Suchepitopes may not be present in vaccines delivering only LPS orO-polysaccharide as the antigen. Furthermore, the conserved sequencesand immunologic cross-reactivity of the Ipa proteins found in allShigella species may enable a vaccine containing the invasins (or otherconserved antigens) to be effective against more than one Shigellaspecies.

The Shigella invasin complex (Invaplex) vaccine prepared from Shigellaflexneri contains the major antigens LPS, IpaB and IpaC and isprotective against homologous challenge in the guinea pigkeratoconjunctivitis and mouse lung challenge models [17, 18]. Afterimmunization, antibodies to LPS, IpaB and IpaC are produced which issimilar to the antibody specificity observed after natural infection inhumans [29]. Additional studies have shown that similar effectiveInvaplex vaccine products can be isolated from all species of Shigellaand EIEC [15]. Although the Invaplex 24 and 50 preparations consist ofmany different proteins, the immunodominant antigens for Invaplex 24 areLPS, IpaB and IpaC and for Invaplex 50 the key antigens are LPS, IpaB,IpaC and the 84 kKa (EF-G) and 72 kKa (DnaK) protein antigens [18].Other proteins within the Invaplex preparations are not immunogenic asdetermined by western blotting techniques using sera collected fromInvaplex-immunized animals. Invaplex 24 and Invaplex 50 can be isolatedfrom all wild-type Shigella species although often-times the Invaplex 24form consistently contains higher quantities of IpaB, IpaC and LPS andfewer non-immunogenic proteins. With the Invaplex vaccine, the highesttiters are often against the immunizing Invaplex (Invaplex 24 orInvaplex 50) when used as an ELISA antigen. This is thought to reflect acomposite response to the Ipa proteins and LPS and to a set ofconformational epitopes preserved by the invasin complex product whenused as an ELISA antigen.

Efforts to identify and purify the active moiety within native Invaplexhas identified a high molecular mass complex that was isolated by sizeexclusion chromatography from native Invaplex 24 preparations. This highmolecular mass complex, referred to as “highly-purified” or HP Invaplex24 consists of predominantly IpaB, IpaC and LPS. HP Invaplex 24 isimmunogenic and protective at levels that are comparable to or exceedthose exhibited by native Invaplex. No other fraction obtained bysize-exclusion chromatography exhibited immunogenic or protectivecapacities comparable to native Invaplex and for this reason the HPInvaplex 24 was considered to be the active moiety within nativeInvaplex responsible for its immunogenicity and efficacy as a vaccine.

Virulent shigellae cause disease by invading, replicating, and spreadingwithin the colonic epithelium by virtue of a complex series of cellularand molecular events orchestrated by an array of plasmid-encodedvirulence factors among which are the Ipa proteins [49]. After invadingthe intestinal epithelial cells of the colonic mucosa a mucosalinflammatory response occurs characterized by an increase inproinflammatory cytokines that leads to recruitment of neutrophils andmacrophages/monocytes. The resulting disease, shigellosis or bacillarydysentery, causes mild to severe diarrhea, fever and intestinal lesions.Since testing of the efficacy of vaccines in humans and nonhumanprimates involves a post-immunization challenge with virulent shigellae,the use of small animal models for initial testing of vaccine candidatesreduces the risk of illness to the human volunteers and primates. Smallanimal models such as the guinea pig keratoconjunctivitis model or themouse lung model of experimental infection with shigellae are largelyused in studies of pathogenesis and preclinical vaccine evaluation [18,44, 50] with the mouse model often used in initial evaluations and theguinea pig model for testing vaccines that are protective in the mousemodel.

The mouse intranasal challenge model of Shigella infection is useful toevaluate Shigella vaccines [18, 31, 51, 52]. The pathogenesis andimmunobiology observed in the pulmonary model parallel those seen in thecolon; that is, virulent Shigella strains invade, replicate, and spreadwithin the epithelium and subsequently elicit antibody as well ascytokine responses [52]. After infection the mice lose weight andultimately die unless protective immunity is present. The ability tomeasure a secretory antibody response, the cellular immune responses,and cytokine responses (largely due to the availability of commercialreagents) makes the mouse model highly attractive for studies on theimmunobiology of shigellosis.

The guinea pig model is an accepted model that is useful for studyingthe virulence of both wild-type and attenuated Shigella strains, and forevaluating the efficacy of potential vaccine candidates [18, 44, 45,53-56]. Several routes of immunization can be employed for immunizationdepending upon the vaccine: oral, intranasal, ocular, and parenteralimmunizations have all been used to protect against ocular challenge.Immunogenicity and efficacy of Shigella vaccine in the guinea pigkeratoconjunctivitis model is now used as a stepping-stone to phase 1clinical studies.

There exists a need for a chemically defined, artificial Invaplexmoiety, which is similar or superior in biological activity to a nativeInvaplex. There is also a need for an Invaplex manufacturing processwhich can be readily scaled up and results in a more specificallydefined product dependent on the ratios of the individual componentparts used. There is also a need for an Artificial Invaplex which iscapable of being designed for specific applications or customizedfunctionality. There is also a need for an Invaplex vaccine that can bemanufactured quickly from its component parts which can be stockpiled inanticipation of future vaccine needs.

BRIEF SUMMARY OF THE INVENTION

The invention relates to an artificial Invaplex and a method for itsmanufacture. The artificial Invaplexes (InvaplexAR) are similar incomposition to HP Invaplex 24. HP Invaplex 24 contains IpaB, IpaC andlipopolysaccharride. The Artificial Invaplex functions as an Invaplexobtained from a native source, though it may have superior activity. TheArtificial Invaplex has defined components: a complex comprising theinvasion proteins IpaB and IpaC, which complex is additionally complexedwith a serotype-specific lipopolysaccharide component from a gramnegative bacteria. The lipopolysaccharide is an immunogen as are IpaBand IpaC. InvaplexAR can function as a mucosal adjuvant and enhance bothserum and mucosal antigen-specific antibody responses as well ascell-mediated immune responses. It is also possible to use otherinvasins from other bacteria species including SipB, SipC or viral (reovirus) proteins associated with endocytosis events or tissue tropisms.

The serotype associated lipopolysaccharides are obtained from gramnegative bacteria such as Shigella flexneri, Shigella sonnei, Shigelladysenteriae, Shigella boydii, enteroinvasive E. coli, Yersinia orSalmonella. The more preferred species include S. flexneri, S. sonnei,S. boydii, S. dysenteriae or enteroinvasive E. coli.

In preparing Artificial Invaplex, e.g. Invaplex_(AR), the invasinproteins used are either highly purified (hp-) or recombinantly (r-)prepared IpaB and IpaC and purified lipopolysaccharride are used asstarting materials. The IpaB and IpaC proteins are mixed to formIpaB:IpaC complex. This complex is mixed with at least onelipopolysaccharide associated with a serotype of a gram negativebacteria to form an Artificial Invaplex. The artificial Invaplex isrecovered from the mixture. Typically, the Artificial Invaplex isremoved based on its charge. Other purification techniques can be used.The order of addition can be varied. The lipopolysaccharide can becomplexed with either of the invasin proteins and then with the otherinvasin protein to form the Artificial Invaplex.

Typically, the amount of IpaC presented relative to IpaB present fallswithin a ratio range of 0.08:1 to 80:1, preferably 0.8:1 to 20:1, morepreferably at least 8:1. It is even more preferred for the amount ofIpaC in the first mixing step to be present in at least a ten foldexcess relative to IpaB. In the second mixing step, thelipopolysaccharide and protein (IpaB and IpaC) are present at a ratiowithin the range 0.01:1 to 10:1, preferably at least 1:2. It is possiblein the second mixing step to include two or more types oflipopolysacchides, which results in an artificial Invaplex beingsuitable for use in multivalent immunogenic compositions, as vaccines.It is also possible in addition to the lipopolysaccharide to include adetectant label, antibiotic, therapeutic or biomolecule comprising anenzyme, protein, polysaccharide, RNA or DNA, or derivatives thereofwhich one wants to incorporate within a cell. This gives rise topossible therapeutic and analytical/diagnostic uses.

The artificial Invaplexes of the invention include those where the IpaCand IpaB are present in a ratio selected from 0.08:1 to 80:1,preferably, 0.8:1 to 20:1, even more preferred approximately 8:1 and thelipopolysaccharride (LPS) is present at a ratio selected from the rangeof 0.01:1 to 10:1, preferably, from 0.5:1 to 5:1, even more preferredapproximately 0.5:1 relative to the total protein (IpaB and IpaC)present. The preferred artificial Invaplexes include S. flexneriInvaplex_(AR) , S. dysenteriae Invaplex_(AR) or S. sonnei Invaplex_(AR)

The artificial Invaplexes of the invention can be formulated to formcompositions suitable for use as immunogenic compositions or vaccines.The Invaplex can be used as the immunogen and/or as the adjuvant. Thecompositions would include in addition to the artificial Invaplex atleast a pharmaceutically acceptable carrier. The amount of Invaplexpresent is an amount effective for the desired function and in at leastthe first instance, is empirically determined. This amount is expectedto be similar to that of a native Invaplex, e.g. Invaplex 24, used for asimilar function.

In addition the artificial invasin complexes facilitate the transport ofa molecule and related materials across cell membranes. These moleculescan be in close proximity to a cell of interest and the artificialInvaplex or can be present within and/or on the artificial Invaplex. Themolecules can be detectant labels, antibiotics, therapeutics, andbiomolecules including proteins, enzymes, RNA, DNA, lipopolysaccharides,polysaccharides and the like.

The artificial Invaplexes can be used in methodologies where nativeInvaplexes have been used, e.g. methods of immunization, methods forfacilitating the transport of biomolecules, e.g. DNA, RNA, proteins,across the membranes of cells, etc. The artificial Invaplex can bedesigned to be multivalent or to have superior activity relative to thenative Invaplex. This should improve the performance of the existingmethod or its utility. Invaplex_(AR) made from recombinant IpaB, IpaCand either S. sonnei LPS or S. flexneri 2a LPS induced immune responsesthat are comparable or higher in magnitude to the immune responseinduced with native Invaplex. IpaB and IpaC responses after immunizationwith Invaplex_(AR) were consistently of higher magnitude than thoseinduced after immunization with native Invaplex. Immunization withInvaplex_(AR) offers comparable or higher levels of protection againstchallenge in the mouse and guinea pig models as compared to immunizationwith native Invaplex. Immunization with Invaplex_(AR) induces cellularimmunity which is of greater magnitude than the Shigella-specificresponse induced with native Invaplex. See, for example, Tables 7 and 8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SDS-PAGE analysis of purified IpaB, IpaC and LPS used toassemble Invaplex_(AR). Purified recombinant IpaB (left) and IpaC(center) were separated by SDS-PAGE and blotted to nitrocellulose (leftpanel for IpaB and IpaC) or stained with coomassie blue (right panel forIpaB and IpaC). Proteins blotted to nitrocellulose were probed overnightwith the anti-IpaB mAb 2F1 or anti-IpaC mAb2G2. S. dysenteriae 1 LPS(right panel) was separated by SDS-PAGE.

FIG. 2 shows purification of S. flexneri 2a (panel A), S. sonnei (panelB) and S. dysenteriae 1 (Panel C) Invaplex_(AR) Purified IpaB, IpaC, andLPS from Shigella species (as indicated) were mixed together at 37° C.for 2 hrs in the presence of 0.5 M NaCl. Purification: after incubation,the mixture was diluted 1/5 and then applied to a HiTrap™ Q HP columnand resolved with a NaCl step gradient as previously described [17].Fractions within each peak were analyzed by 13% SDS-PAGE. TheInvaplex_(AR) (red peak) eluted in the 0.5 M NaCl (50% Buffer B) step.Gels were either stained with Coomassie Blue (gel C), blotted tonitrocellulose and probed with mAbs specific for IpaB and IpaC (gel W)or reacted with silver (gel S). Purified LPS from the designatedShigellaspecies is in the left lane of the “S” panel. The 0.5M NaCl stepcontained the majority of IpaB, IpaC and LPS; the 0.24 M NaCl peakcontained small quantities of IpaB, IpaC and LPS.

FIG. 3 shows SDS-PAGE (coomassie blue, western blots and silver stains)of components used to make InvaplexAR for S. dysenteriae 1 and the finalInvaplex_(AR) product. The gel on the left is a coomassie blue stainedgel of purified IpaB, IpaC and two different lots of S. dysenteriae 1Invaplex_(AR). The prominence of IpaB and IpaC are evident in theInvaplex_(AR). The middle panel is a western blot that used mAbs to IpaBand IpaC to probe the purified proteins and the two different lots of S.dysenteriae 1 InvaplexAR. The panel on the right is a silver-stained gelof the purified IpaB and IpaC proteins, purified LPS, and two lots of S.dysenteriae 1 Invaplex_(AR) which show both the Ipa protein bands andthe LPS bands in the InvaplexAR.

FIG. 4 shows detection of Internalized native and artificial S.dysenteriae 1 Invaplex in mammalian cells. BHK-21 cells were incubatedat 37° C. for 60 min with LPS (A), native Invaplex 24 (B), orInvaplex_(AR) (C) all produced from S. dysenteriae-1. Fixed cells wereprobed with anti-S. dysenteriae 1 polyclonal sera followed byGAM-IgG-OG₄₈₈ to visualize internalized Invaplex (green). Cells werecounterstained with DAPI to identify the nucleus (blue). Shigella LPSwas not internalized by the host cells whereas both native Invaplex andartificial Invaplex were internalized by ˜70% of cells.

FIG. 5 shows Invaplex 50, Invaplex 24 and LPS-specific serum IgG and IgAend point titers on day 42 after intranasal immunization with S.flexneri 2a native Invaplex 24, Invaplex_(AR), purified IpaB, IpaC or S.flexneri 2a LPS.

FIG. 6 shows Anti-IpaB and IpaC serum IgG endpoint end point titers onday 24 after intranasal immunization with S. flexneri 2a native Invaplex24, Invaplex_(AR), purified IpaB, IpaC or S. flexneri 2a LPS.

FIG. 7 shows antigen-specific IgA in intestinal washes collected on day35 from mice intranasally immunized with native S. flexneri 2a Invaplexor Invaplex_(AR). Data represents the mean endpoint titer plus onestandard error of the mean for each group of mice (n=4 mice/grp).

FIG. 8 shows antigen-specific IgA in lung washes collected on day 35from mice intranasally immunized with native S. flexneri 2a Invaplex orInvaplex_(AR). Data represents the mean endpoint titer plus one standarderror of the mean for each group of mice (n=4 mice/grp).

FIG. 9 shows Invaplex-specific serum IgG and IgA endpoint titers in miceafter intranasal immunization with OVA alone, or OVA combined withInvaplexAR, native Invaplex, or cholera toxin. Groups of mice wereintranasally inoculated on day 0, 14, and 28 with OVA or OVA combinedwith either Invaplex_(AR), Invaplex or CT. Blood collected on day 0, 28,and 42 were analyzed by ELISA for anti-Invaplex serum IgG and IgAendpoint titers. Data represents the mean (n=5 mice/grp) endpointtiter±1 SEM.

FIG. 10 shows S. flexneri 2a LPS-specific serum IgG and IgA endpointtiters in mice intranasally immunized with OVA alone, OVA combined withInvaplexAR, native Invaplex, or cholera toxin. Groups of mice wereintranasally inoculated on day 0, 14, and 28 with OVA or OVA combinedwith either Invaplex_(AR), Invaplex, or CT. Blood collected on day 0,28, and 42 were analyzed by ELISA for anti-Invaplex serum IgG and IgAendpoint titers. Data represents the mean (n=5 mice/grp) endpointtiter±1 SEM.

FIG. 11 shows Shigella invasion-specific serum IgG and IgA endpointtiters in mice intranasally immunized with OVA alone, or OVA combinedwith InvaplexAR, native Invaplex or cholera toxin. Groups of mice wereintranasally inoculated on day 0, 14, and 28 with OVA or OVA combinedwith either Invaplex_(AR), Invaplex or CT. Blood collected on day 0, 28,and 42 were analyzed by ELISA for anti-IpaB and IpaC serum IgG endpointtiters. Data represents the mean (n=5 mice/grp) endpoint titer±1 SEM.Comparisons between groups were accomplished using two-way ANOVAanalysis of natural log-transformed endpoint titers.

FIG. 12 shows ovalbumin-specific serum IgG and IgA endpoint titers inmice intranasally immunized with OVA alone, or OVA combined withInvaplexAR, native Invaplex or cholera toxin. Groups of mice wereintranasally inoculated on day 0, 14, and 28 with OVA or OVA combinedwith either Invaplex_(AR), Invaplex, or CT. Blood collected on day 0,28, and 42 were analyzed by ELISA for anti-OVA serum IgG and IgAendpoint titers. Data represents the mean (n=5 mice/grp) endpointtiter±1 SEM. Comparisons between groups were accomplished using two-wayANOVA analysis of natural log-transformed endpoint titers.

FIG. 13 shows S. flexneri 2a LPS, Invaplex 24 and ovalbumin-specificlung IgG and IgA endpoint titers in mice intranasally immunized with OVAalone, or OVA combined with InvaplexAR, native Invaplex or choleratoxin. Groups of mice were intranasally immunized on day 0, 14, and 28with OVA or OVA combined with either InvaplexAR, Invaplex, or CT. Lungwashes were collected from individual mice on day 42 and analyzed byELISA for anti-LPS, anti-Invaplex 24, and anti-OVA IgG and IgA endpointtiters. Data represents the mean (n=5 mice/grp) endpoint titer±1 SEM.

DETAILED DESCRIPTION OF THE INVENTION Biochemistry of ArtificialInvaplex Production and Purification of Recombinant IpaB and IpaC andnative S. flexneri, S. sonnei and S. dysenteriae 1 LPS Purification ofIpa Proteins

The Ipa proteins are highly conserved in all Shigella spp [32, 33].Recombinant E. coli expressing either IpaB or IpaC have been previouslydescribed. Two strategies are available for purifying recombinant Ipaproteins. Histidine-tagged (HisTag) recombinant IpaC is purified byaffinity chromatography using nickel columns. The effect of thehistidine residues on the Ipa protein's biological or immunologicalfunction or the ability of the proteins to form Invaplex is not knownbut HisTag-IpaC appears to maintain biological activity [30]. Analternative purification procedure has been used to produce recombinantIpaB. This method takes advantage of IpaB's native affinity for thechaperone IpgC [34, 35]. By co-expressing IpaB together with theHisTag-chaperone IpgC in the same recombinant organism, it will bepossible to purify the IpaB/chaperone complex. After purification, theHisTag-IpaC/IpaB protein complex is denatured resulting in monomericIpaB protein and HisTag-IpgC which allows the HisTagged chaperone to beselectively removed on a nickel column while the free Ipa protein runsthrough the column.

Purification of HisTag-IpaC

The recombinant IpaC was expressed in an E. coli background using anIPTG inducible expression system (pET plasmid). Specifically, the IpaCgene was cloned into the pET15b plasmid (Novagen) and expressed in theE. coli vector BL21(DE3)pLysS [30]. The recombinant IpaC protein hasseveral histidine residues on the amino terminal end that allowssubsequent purification on a nickel column [30]. The IpaC proteinexpressed in the recombinant is located in inclusion bodies and requiressolubilization with urea. Removing the urea often leads to insolubilityif the IpaC concentration is too high (>1 mg/ml). Thus, IpaC eluted fromthe nickel column was maintained in urea to maintain solubility. Usingsmall-scale cultures (3 L) it has been possible to produce approximately75 mg of purified IpaC with a yield of approximately 8 mg/L. The productis greater than 90% pure and is reactive with anti-IpaC mAb 2G2 [32](FIG. 1). The purified protein is soluble and can be stored frozen. Anexample of purified IpaC is in FIG. 1.

Purification of IpaB

The recombinant organism expressing IpaB is in an E. coli BL21(DE3)background. The IpgC gene was cloned into pET15b; the IpaB gene wascloned separately into the pACYC-duet vector. After IPTG induction theHisTag IpgC/IpaB complex is solubilized and purified on a nickelaffinity column. The HisTag-IpgC/IpaB complex is released with EDTA andprepared for second application to the nickel affinity column by removalof the EDTA and the addition of the non-ionic detergent OPOE(N-octyl-oligo-oxyethylene) to a final concentration of 1% v/v. The OPOEwill disrupt the HisTag-IpgC/IpaB complex thereby allowing the freeHisTag-IpgC protein to bind to the nickel (Ni-Sepharose) column and theuntagged, free IpaB to flow through the column for collection. Fractionsfrom the void volume were probed for IpaB with anti IpaB mAb 2F1.Positive fractions were analyzed by SDS-PAGE (stained) to determine ifcontaminating HisTag-IpgC was present. If so, the material was treatedagain with OPOE and reapplied to the nickel column to remove residualHisTag-IpgC. The resultant soluble IpaB product has little to noHisTag-IpgC “contamination”, is over 90% pure and reacts with anti IpaBmAb 2F1 [32] (FIG. 1). The yield of IpaB per liter of starting culturewas approximately 3.5 mg/L.

Purification of S. flexneri 2a , S. sonnei, or S. dysenteriae 1 LPS

S. flexneri 2a, S. sonnei, and S. dysenteriae 1 LPS were produced by theWestphal procedure [36] which involves a hot phenol/water extraction ofthe shigellae. Virulent or attenuated strains of Shigella can be used assource of LPS as long as the smooth LPS phenotype is expressed. Inexperiments described below, wild-type S. flexneri 2a (strain 2457T) andS. sonnei (Mosely) were used. For S. dysenteriae 1, the attenuatedstrain WRSd1 was used to minimize risk of infection to laboratorypersonnel. WRSd1 is a virG, stx knockout previously produced at WRAIR[37].

Extraction, Purification and Characterization of LPS

LPS is extracted by the Westphal procedure [36]. Briefly, the bacterialcell pellets are suspended in hot (68° C.) distilled water (5 ml waterfor each gram of pellet). An equal amount of phenol, heated to 68° C.,is added and the pellet solution is vigorously shaken for 15 minutes.The bottles are then cooled to approximately 10° C.±5° C. The samplesare centrifuged, the aqueous phase removed and stored at 4° C.Extraction of the cell mass is performed a second time and all aqueousphases are pooled. The aqueous phase is dialyzed against distilled waterfor two days, and then centrifuged (8000×g, 30 min) to remove extraneouscellular debris. This supernatant is subjected to ultracentrifugation(90,000×g) for 2 hours and the pellet is saved. The pellet is rinsedwith sterile distilled water, resuspended in sterile distilled waterovernight at 4° C., pooled and lyophilized. The final lyophilizedproduct is weighed and then a small portion (<10 mg) is removed,dissolved in 1 ml of endotoxin-free water and characterizedbiochemically (see below).

Endotoxin content of the purified LPS is performed by the chromogenicLAL. E. coli endotoxin serves as a control reagent for this analysis.All results are reported in terms of international endotoxin units (EU).The purified LPS is also analyzed by SDS-PAGE with silver stain todetermine if the typical multiple band profile of smooth LPS arepresent. FIG. 1 shows a silver stained gel of the purified S.dysenteriae 1 LPS demonstrating the typical multiple banding pattern ofsmooth LPS. The final LPS product has residual amounts of protein (<5%,determined by Bradford assay) and DNA (<5%, determined by Hoechst stain)and is reactive with LPS serotype specific antibodies (anti-S.dysenteriae 1 LPS mAb MAB753 (Chemicon International) for S. dysenteriae1 LPS; mAb 2E8 for S. flexneri 2a LPS; MAB755 (Chemicon) for S. sonneiLPS) by western blot or ELISA.

Quantitation of IpaB and IpaC Content in Invaplex_(AR) by Immunoassay

The amount of IpaB and IpaC in Invaplex (artificial or native) wasdetermined using a modified ELISA procedure. The ELISA used purifiedrecombinant IpaB or IpaC proteins to generate standard curves fordetermination of the quantity of the antigens in the Invaplexpreparations. Immulon 1B ELISA plates (ThermoLab Systems) were coatedovernight at 4° C. with either 50 μl of recombinant IpaB, recombinantIpaC, or Invaplex_(AR). Antigen was titrated (in triplicate) using2-fold serial dilutions in carbonate coating buffer (0.2 M carbonate, pH9.8) with starting concentrations of 125 ng/ml (IpaB), 200 ng/ml (IpaC),and 10 ug/ml (Invaplex). After washing and blocking with casein,affinity-purified monoclonal antibodies specific for IpaB (2F1) or IpaC(2G2) [32] were incubated with the antigen-coated plates for 2 hours.After washing, antigen-specific antibody was detected using anti-mouseimmunoglobulin G (IgG) conjugated with alkaline phosphatase (Kirkegaard& Perry). Using the substrate para-nitrophenyl phosphate the opticaldensity at 405 nm (OD₄₀₅) was measured using an ELISA plate reader(Molecular Devices, Menlo Park, Calif.) after a 60 minute incubationwith substrate. Using the Softmax Pro 4.5 (Molecular Devices) program, astandard curve plotting OD₄₀₅ versus concentration (ng/ml) wasdetermined. The concentration of the unknown samples were theninterpolated from the standard curve.

Invaplex_(AR) is designed to have IpaB and IpaC concentrations and anIpaC/IpaB ratio that is similar to HP Invaplex 24. The ratio of thequantity of IpaC to IpaB was determined for the Invaplex_(AR) andcompared to HP-Invaplex 24, the most pure form of Invaplex. TheIpaC/IpaB molar ratio in HP Invaplex is approximately 8. This wasdetermined by densitometry analysis of SDS-PAGE gels and quantitativeantibody based assays for IpaB and IpaC. In addition the LPS content isexpected to be at the same relative mass ratio (approximately 0.5 to 0.6mg of LPS for every 1 mg of protein) that is found in HP-Invaplex.

Measurement of LPS in Invaplex

LPS content in Invaplex preparations was measured by determining theamount of 2-keto-3-deoxyoctonate (KDO) in each preparation [43] or byusing the limulus amoebocyte lysate assay (Cambrex Inc.).

Method for Formation of Artificial Invaplex. Preparation ofInvaplex_(AR) for S. flexneri 2a, S. sonnei and S. dysenteriae 1

The purified components were mixed at ratios similar to that found inhighly purified native Invaplex to form the artificial Invaplex.Analysis of the S. flexneri 2a HP Invaplex indicated that the IpaC/IpaBmolar ratio was approximately 8.0 and the LPS to total protein ratio wasapproximately 0.56 mg LPS/mg total protein. Using these parameters as aguide for reconstituting Invaplex from purified IpaB, IpaC and LPS aseries of experiments were conducted to create an Invaplex_(AR). Onceformed, the artificial Invaplex was purified by ion-exchange FPLC.

Purified, soluble IpaB and IpaC, each in their respective final bufferswere mixed together at an IpaC/IpaB molar ratio of 8. After the IpaB andIpaC were mixed, the solution was slowly added to dry LPS powder (ratioof LPS to total protein is 0.56). LPS from any Shigella species can beused; for the described experiments S. flexneri 2a, S. sonnei or S.dysenteriae 1 LPS was used. The mixture was incubated at 37° C. forapproximately 2 hours with shaking. Afterwards the protein/LPS mixturewas diluted to 20 mM Tris-HCl, 0.10 M NaCl and 1.2 M urea to reduce theNaCl concentration prior to ion-exchange chromatography. The dilutedmixture was then purified on a HiTrap™ Q HP anion exchange column.

Assembly Experiments: Formation of a S. flexneri Invaplex_(AR)

Preliminary assembly experiments accommodated the insolubility of IpaCby maintaining both IpaC and IpaB proteins in a buffer containing ≧4Murea. Once the IpaC/IpaB mixture was added to LPS, solubility was nolonger an issue which permitted the eventual removal of the urea.Typically purified IpaC by itself will precipitate upon dilution. Inpreliminary mixing experiments the 8 IpaC/IpaB molar ratio wasmaintained by adding 8 μM HisTagIpaC to 1 μM IpaB in a final volume of 1ml. Both proteins were initially prepared in 20 mM Tris-HCl, 0.5 M NaCl,6 M urea, pH 7.9. The proteins were mixed in a glass test tube andincubated at 37° C. without shaking for 15 mins. Dry LPS (230 μg) in aseparate glass tube was also incubated at 37° C. without shaking for 15mins. After the 15 min incubation, the IpaB+IpaC mixture was used tosolubilize the pre-warmed LPS by slowly adding the protein mixture downthe side of the tube, followed by vortexing. No appreciable flocculationor precipitation was observed. The IpaB/IpaC/LPS mixture was thenincubated at 37° C. with shaking (200 rpm) for 2 hrs. In preparation forion exchange chromatography (IEC), the mixture was diluted five-foldwith pre-warmed 20 mM Tris buffer, pH 9.0 to lower the saltconcentration. No precipitation was observed as the mixture cooled toroom temperature. For final purification, the diluted IpaB/IpaC/LPSmixture was applied to an anion exchange column (HiTrap™ Q HP) withBuffer A consisting of 20 mM Tris-HCl, pH 9.0 and Buffer B consisting of1 M NaCl, 20 mM Tris-HCl, pH 9.0, and a step gradient of 0% (5 columnvolumes) to 24% (10 column volumes) to 50% (6 column volumes) to 100%Buffer B (5 column volumes). One ml fractions were collected from a 1 mlHiTrap™ Q HP column at an elution flow rate of 1 ml/min. Fractions fromeach step were analyzed for the presence of IpaB, IpaC and LPS byspotblot. The S. flexneri Invaplex_(AR) eluted in the 50% B step (FIG.2) which contained the greatest quantities of all three components(IpaB, IpaC and LPS) as determined by western blots and silver stainedgels (FIG. 2A). When applied to a size exclusion column theIpaB/IpaC/LPS complex eluted at between the 1 MDa and 669 kKa standardswhich is the same size range as HP Invaplex.

Formation of S. dysenteriae 1 Artificial Invaplex and Larger ScaleProduction

The above experiment was repeated but with a ten-fold increase inreactants to increase the Invaplex_(AR) yield. In addition S.dysenteriae 1 LPS was used instead of S. flexneri LPS. As such, 3.28 mgof IpaC in 20 mM Tris-HCl, 0.5 M NaCl, 6 M Urea, pH 7.9, was mixed with0.62 mg IpaB in 20 mM Tris-HCl, 0.5 M NaCl, pH 7.9, in a total volume of5 mls. The protein mixture and a separate tube of LPS (2.1 mg) waspre-warmed to 37° C. The IpaB+IpaC mixture was added to the LPS tube andvortexed to solubilize the LPS. No precipitation was observed. Thereaction mixture was incubated at 37° C. with shaking (200 rpm) for 2hours. Next pre-warmed IEC Buffer A (20 mM Tris-HCl, pH 9.0) was addedto dilute the salt concentration 1:5 yielding a final volume of 25 mls.After cooling to room temperature, the diluted reaction mixture wasapplied to a 1 ml HiTrap™ Q HP IEC column and subjected to an Invaplexpurification step gradient (see above). The S. dysenteriae 1Invaplex_(AR) eluted in the 50% Buffer B step. It contained IpaB, IpaCand S. dysenteriae 1 LPS. (FIG. 2C). Analysis of the 0.24 M and 1.0MNaCl peaks did not detect significant quantities of IpaB, IpaC or LPS.The yield of Invaplex_(AR) production was approximately 10 to 20% of theamount of total protein used to initiate the assembly process.

Similar assembly experiments have been conducted for S. sonnei resultingin an InvaplexAR for S. sonnei (see FIG. 2B).

Large Scale Production of Invaplex_(AR) of Shigella flexneri 2a

A fifty-fold increase in reactants was used to produce moreInvaplex_(AR) for advanced studies. Using an 8 IpaC/1 IpaB molar ratio,16.4 mg of IpaC (in 20 mM Tris-HCl, 0.5 M NaCl, 6 M Urea, pH 7.9) wasmixed with 3.1 mg IpaB (in 20 mM Tris-HCl, 0.5 M NaCl, pH 7.9) in atotal volume of 20 mls. The protein mixture and a separate tube ofShigella flexneri 2a LPS (11.4 mg) were pre-warmed to 37° C. TheIpaB+IpaC mixture was added to the LPS tube and vortexed to solubilizethe LPS. The reaction mixture was incubated at 37° C. with shaking (200rpm) for 2 hours. Next pre-warmed IEC Buffer A (20 mM Tris-HCl, pH 9.0)was added to dilute the salt concentration 1:5 yielding a final volumeof 100 mls. After cooling to room temperature, the diluted reactionmixture was applied to a 5 ml HiTrap™ Q HP IEC column and subjected toan Invaplex purification step gradient (see above). The S. flexneri 2aInvaplex_(AR) eluted in the 50% Buffer B step. It contained IpaB, IpaCand S. flexneri 2a LPS. Analysis of the 0.24 M and 1.0M NaCl peaks didnot detect significant quantities of IpaB, IpaC or LPS. The yield ofInvaplex_(AR) production was approximately 9% of the amount of totalprotein used to initiate the assembly process.

Analysis of Invaplex_(AR) Preparations by Electrophoresis and WesternBlots for IpaB and IpaC

The total protein composition of the Invaplex_(AR) preparations wasdetermined by stained SDS-PAGE gels. Western blots were used todetermine the presence, size and relative concentrations of IpaB andIpaC. Silver stained gels were used to assess the LPS profile [18].Monoclonal antibodies specific for either the IpaB or IpaC proteins wereused to probe the nitrocellulose blots [32]. After probing with alkalinephosphatase-conjugated protein A, blots are developed with ASMXphosphate (Sigma) and Fast Red TR salt (Sigma). Video images of theblots are displayed on a Macintosh computer using a CCD video camera(Cohu) and captured with NIH Image software (version 1.62). Densitometryanalysis of the digital images were performed with GelPro Image analysissoftware (version 2.0.10, Media Cybernetics, Inc.). The total proteingel profile and the IpaB and IpaC content should resemble that found inHP-Invaplex 24 or HP-Invaplex 50.

Size Exclusion Chromatography

Size-exclusion chromatography was used to determine if the components ofInvaplex_(AR) found in the ion-exchange peak are truly complexed. Thisis the same method used to produce HP-Invaplex. A Superose 6 HR 10/30column (Amersham Pharmacia), calibrated with Blue Dextran 200 (2 MDa),thyroglobulin (669 kKa),and ovalbumin (43 kKa), was loaded withInvaplex_(AR) (approximately 3 mg). Fractions (0.5 ml, flow rate 0.25ml/min) were collected and analyzed by immuno-spot blot for the presenceof IpaB, IpaC and LPS. All three Invaplex_(AR) components (IpaB, IpaCand LPS) eluted in fractions between 2 MDa and 669 kKa indicating theyare associated in a complex as they eluted at a size much greater thanthe individual components (43 kKa for IpaC; 62 kKa for IpaB). The SECsize of Invaplex_(AR) is similar to the size where HP-Invaplex elutes.

Evaluation of S. dysenteriae 1 Invaplex_(AR) in Tissue Culture model ofInternalization

Invaplex binds to and stimulates endocytosis in cultured mammaliancells. This property is dependent on the presence of the invasins IpaBand IpaC and utilizes host-cell acting during the endocytic process[38]. The Invaplex internalization assay is an in vitro model used tomeasure the functional activity of Invaplex preparations. The assay ismodeled after the Shigella plaque assay [39]and Shigella uptake assays[40, 41]. The assay involves incubating Invaplex_(AR) with BHK-21fibroblast cells (60-70% confluent) in chamber slides. After incubationfor 5, 15, 30, or 60 minutes the cells were washed 3× with PBS andformalin-fixed. Fixed cells were permeabilized with PBS supplementedwith 0.1% Triton X-100 and 0.1% BSA (wash buffer) and probed withpolyclonal mouse serum with specificity for LPS, IpaB, and IpaC. Boundpolyclonal mouse antibodies were detected with GAM-IgG-Oregon Green 488(Molecular Probes) followed by counterstaining with DAPI to identify thenuclei. The Invaplex treated cells were examined by fluorescentmicroscopy with digital image capturing. Both native and artificial S.dysenteriae 1 Invaplex were internalized by BHK cells with similarcytoplasmic staining pattern for both preparations indicating that theInvaplex_(AR) maintains the capacity to stimulate internalization bymammalian cells (FIG. 4). This indicates that the assembly ofrecombinant IpaB and IpaC with S. dysenteriae 1 LPS into a complexisolated by ion-exchange FPLC has resulted in a synthetic product whichmaintains the same biological activity as the native complex.

Immunogenicity, Adjuvanticity and Protective Efficacy of Native andArtificial Invaplex Vaccines

The mouse lung-infection [31] and the guinea pig keratoconjunctivitsmodels [44] were used to determine whether intranasal immunization withS. flexneri 2a Invaplex_(AR) stimulates a protective immune responsethat is effective against a homologous S. flexneri 2a challenge.Separate mouse studies were used to determine whether immunization withS. sonnei Invaplex_(AR) could protect against a homologous S. sonneichallenge and against a heterologous challenge with S. flexneri 2a.Additional studies were conducted to assess the adjuvant activity ofInvaplex_(AR) using ovalbumin (OVA) as an immunogen. In theadjuvanticity experiments, immune responses induced after immunizationwith OVA alone were compared to immune responses induced with OVAcombined with either native Invaplex, Invaplex_(AR), or cholera toxin,an established, potent mucosal adjuvant. The guinea pig and mouseexperiments were conducted at WRAIR under IACUC approved protocols.

Mouse Experiments

Four separate animal experiments have been completed to evaluate theimmunogenicity, adjuvanticity, and protective efficacy of artificialInvaplex. Common methods were used in each experiment and are outlinedin the following paragraph. Methods and materials specific to each studyare indicated in separate sections.

Immunizations and Challenge

Mice were immunized on days 0, 14 and 28. A total antigen volume of 25ul was delivered in 5 to 6 small drops applied to the external nareswith a micropipette. Blood taken by tail bleed from all mice at days 0,28 and 42 and 2 weeks post-challenge (day 63). On day 35 or 42 mice wereeuthanized followed by immediate collection of mucosal washes andremoval spleen for in vitro proliferation and cytokine analysis S.flexneri 2a Invaplex_(AR). Three weeks after the final immunization mice(15 per group) were challenged intranasally with a lethal dose (1.6×10⁷cfu) of S. flexneri 2a (2457T) or with S. sonnei (Mosely, 8×10⁶ cfu) asdescribed for the mouse lung model [34]. Prior to intranasalimmunization or challenge, mice are anesthetized with a mixture ofketamine hydrochloride (40 mg/kg) and xylazine (12 mg/kg). Challengedmice were monitored daily for two weeks for weight loss, fur rufflingand lethargy, with death used as an endpoint. Surviving mice were bled14 days post-challenge (day 63).

S. flexneri 2a Invaplex_(AR) Immunogenicity and Protection Study I

Separate groups of female Balb/c mice (10-15 mice/group) were immunizedintranasally on day 0, 14, and 28 with 5 ug of either the native S.flexneri 2a Invaplex (lot JWJX) or 2.5 μg of artificial S. flexneri 2aInvaplex_(AR) (Lot KF-D3D4) vaccines. Control animals were inoculatedwith saline. Blood was collected on day 0, 28, 42, and 63. Animals werechallenged intranasally on day 49.

S. flexneri 2a Invaplex_(AR) Immunogenicity and Protection Study II

Separate groups of female Balb/c mice (15-20 mice/group) were immunizedintranasally on day 0, 14, and 28 with 5 ug of either the native S.flexneri 2a Invaplex (Lot JWJX) or 2.5 μg of artificial S. flexneri 2aInvaplex_(AR) (lot KR-C5) vaccines. Control animals were inoculated withsaline. Blood was collected on day 0, 28, 42, and 63. Lung andintestinal washes were collected on day 42 from 4 animals/group.Remaining animals were challenged intranasally on day 49.

S. flexneri 2a Invaplex_(AR) Adjuvanticity Study

The ability of Invaplex_(AR) to function as a mucosal adjuvant wasdetermined in mice using ovalbumin (OVA) as a model protein antigen.Balb/cByJ mice (5 mice/group) were intranasally immunized on day 0, 14,and 28 with OVA (5 μg) alone, or OVA (5 μg) combined with either S.flexneri 2a Invaplex_(AR) (lot KF-D3D4; 2.5 μg), native S. flexneri 2aInvaplex (Lot JWJX; 5 μg), or cholera toxin (CT; 5 μg). Blood wascollected on day 0, 28, and 42. Control animals were inoculated withsaline. Lung and intestinal washes were collected on day 42. Serumendpoint titers on day 0, 28 and 42 with specificity for Invaplex 24,Invaplex 50, LPS, OVA, IpaB and IpaC were determined by ELISA.Antigen-specific antibodies in mucosal washes were also assessed.

S. sonnei Invaplex_(AR) Immunogenicity and Protection Study

The immunogencity and protective efficacy of S. sonnei Invaplex_(AR) wasdetermined in mice using a homologous (S. sonnei 53G) and heterologous(S. flexneri 2a 2457T) challenge. Balb/cByJ mice (10-15 mice/group) wereintranasally immunized on day 0, 14, and 28 with S. sonnei Invaplex_(AR)(lot KJ-D3D6; 2.5 μg), native S. sonnei Invaplex (Lot JOJP; 5 μg), ornative S. flexneri 2a Invaplex (Lot JWJX; 5 μg). Control animals wereinoculated with saline. Blood was collected on day 0, 28, and 42.

Guinea Pig Experiments

Guinea pigs (Hartley strain, 6 to 10 per group) were immunizedintranasally with the artificial or native Invaplex vaccines (25ug/dose). The dose volume (100 ul) was split equally between eachnostril. Saline was used to immunize control animals. Guinea pigs wereimmunized on days 0, 14, and 28 and bled from the lateral ear vein ondays 0, 28, 42, and 14 days after challenge. Prior to intranasalimmunization, guinea pigs were anesthetized with a mixture of ketamine(40 mg/kg) and xylazine (4 mg/kg). Three weeks after the thirdimmunization guinea pigs were challenged intraocularly with S. flexneri2a (strain 2457T) and observed daily for 5 days to assess the degree ofinflammation and keratoconjunctivitis as previously described [44].

ELISA

ELISA assays are used to measure antigen specific IgA and IgG antibodiesin sera and mucosal secretions, including lung and intestinal lavagesand stool extracts from immunized and/or challenged mice and guinea pigs[18, 29, 46]. Antigens used in ELISAs, including purified S. flexneri 2aLPS, S. sonnei LPS, water-extracted Shigella antigens, purified IpaB andIpaC proteins, and S. flexneri 2a native Invaplex and OVA were coatedonto ImmunIon IB 96-well ELISA plates (ThermoLab Systems) overnight at4° C. After blocking with 2% casein (in Tris-saline buffer, pH 7.5) seraand mucosal washes were serially diluted in duplicate and incubated withthe antigen-coated plates for 4 hours at 25° C. After washing with PBScontaining 0.05% Tween 20, antigen-specific antibody is probed withalkaline phosphatase conjugated anti-mouse IgG or anti-mouse IgA(Kirkegaard & Perry). Alkaline phosphatase activity was detected withp-nitrophenyl phosphate (1 mg/ml). After a 30-min incubation insubstrate, the optical density (OD₄₀₅) was measured using an ELISA platereader (Molecular Devices, Menlo Park, Calif.). Endpoint titers weredetermined for each animal and was used to calculate geometric meantiters for each group at each time point. Typically animals intranasallyimmunized with Invaplex respond with a 4 to 8-fold higher serum (IgG andIgA) and mucosal (lung and intestine) IgA titers to Shigella antigens ascompared to preimmune samples or saline control animals.

Total IgA Assay

A capture enzyme immunoassay was used to determine total IgAconcentrations in mucosal samples. Sample concentrations were determinedfrom standard curves, using purified mouse IgA assayed in parallel.Specific mucosal IgA activities were calculated by dividing the endpointtiter for each individual mucosal sample by the concentration of totalIgA within the same sample [47].

Antigen Stimulation of Cultured Lymphocytes from Mice Immunized withInvaplex_(AR)

Lymphocyte proliferation upon antigen stimulation was determined usingsplenocytes collected from Invaplex-immunized mice and naive mice.Mononuclear cells (2×10⁵ per well) were incubated with Invaplex, IpaB,or IpaC or S. flexneri 2a LPS preparations. Simultaneously in separatemicrotiter wells, splenocytes were stimulated with concanavalin A toprovide positive controls. Negative controls included cells incubatedwith complete medium alone and cells from naive mice stimulated withantigen. After incubation with antigen for 4 to 7 days, cellproliferation was measured by a non-radioactive assessment ofdehydrogenase activity using MTS (Promega) [48]. There is a strongcorrelation with increasing optical density readouts in this assay andthe number of viable cells in a well. Prior to measuring proliferation,cell culture supernatants were collected on days 3 and 5 for cytokinemeasurements (see below). Stimulation indices (SI) were calculated bydividing the mean optical density of antigen-stimulated cells by themean optical density of medium-only stimulated cells. The SI of cellsfrom mice immunized with Invaplex was compared to the SI of cells fromnon-immunized mice.

Antigen-Specific Systemic Antibody Response after IntranasalImmunization with Invaplex_(AR)

The immunogenicity of artificial S. flexneri 2a Invaplex(Invaplex_(AR)), manufactured from individual purified components ratherthan the virulent organism (native Invaplex) was evaluated in mice.Groups of mice (n=6-10) were intranasally immunized on day 0, 14, and 28with native S. flexneri 2a Invaplex 24 (5 or 10 μg), Invaplex_(AR) (2.5μg), purified IpaB (2.5 μg), purified IpaC (2.5 μg), LPS (2.5 μg), orsaline. Three weeks after the third immunization (day 49), the mice werechallenged with Shigella flexneri 2a (2457T). Blood collected on day 0,28, 42, and 63 was analyzed by ELISA for serum IgG and IgA responses toInvaplex 50, Invaplex 24, purified LPS, IpaB and IpaC. FIGS. 5 and 6outline the serum IgG and IgA endpoint titers determined in bloodcollected on day 42 (two weeks after the third immunization).

Saline-inoculated mice and preimmune sera from immunized mice did nothave detectable levels of antigen-specific antibodies (data not shown).Immunization with S. flexneri 2a Invaplex_(AR) induced serum IgG and IgAresponses to Invaplex 50 and Invaplex 24 of comparable magnitude tothose induced with native Invaplex 24 (FIG. 5), and significantly higher(p<0.001) than those induced after inoculation with saline. A two-foldincrease in the amount of native Invaplex (lot JWJX) used forimmunization did not result in an increase in the magnitude of theInvaplex 50, LPS, or Invaplex 24-specific serum IgG or IgA responsesmeasured on day 42 (FIG. 5). Immunization with purified IpaB resulted ina strong IpaB-specific (FIG. 6) and Invaplex 24-specific response(GMT>5760 and 3800, respectively) and a moderate response to Invaplex 50(GMT 950). Immunization with purified LPS did not induce a detectableserum IgG or IgA response to any of the antigens used in ELISA in themajority (5/6) of mice in the vaccine group. Similarly, immunizationwith purified IpaC also did not induce a detectable immune response toany of the antigens assayed, including a IpaC-specific ELISA.Interestingly, the mice immunized with Invaplex_(AR) had among thehighest IpaC-specific endpoint titers (FIGS. 6 and 8, significantlyhigher (p<0.001) than those induced after immunization with nativeInvaplex.

Protection of Mice from a Lethal Challenge of S. flexneri afterIntranasal Immunization with Invaplex_(AR)

The lethal lung model entails intranasal inoculation of mice with alethal dose of shigellae which establish an infection of the lungsleading to severe weight loss, pneumonia and ultimately death over theobservation period of two weeks. Three weeks after intranasalimmunization with S. flexneri 2a Invaplex_(AR) or native Invaplex micewere challenged with a lethal dose of S. flexneri 2a (strain 2457T). Innaive mice (treated with saline) 11 of 13 mice died with a mean maximumweight loss of 31.4% of the pre-challenge weight (see Table 1). All miceimmunized with S. flexneri 2a Invaplex_(AR) (p<0.001) or native Invaplex(p<0.001) survived the lethal challenge with S. flexneri 2a. Withrespect to weight loss (which is likely a more sensitive indicator ofprotective immunity) mice immunized with Invaplex_(AR) lost less oftheir pre-challenge weight (21.3%) as compared to 23.8% to 26.0% weightloss in the native Invaplex-immunized mice. Furthermore the day ofweight rebound (an indication of recovery from the challenge) was day 7for Invaplex_(AR) and day 13 (native Invaplex, 5 μg) or day 7 (nativeInvaplex, 10 μg).

In addition the protective capacity of the individual components (IpaB,IpaC and LPS) used to construct Invaplex_(AR) were also evaluated in themouse lethal lung model (see Table 1). Immunization with purified LPSand purified IpaC did not protect mice from death (both P>0.05) andalthough immunization with IpaB protected 5 of 6 mice from a lethalchallenge the mice never regained weight and remained symptomatic (lowweight, ruffled fur) through the end of the observation period. The meanmaximum weight loss after challenge was 29.9% for LPS, 31.0% for IpaBand 33.9% for IpaC all of which are very close to the naïve mice weightloss value of 31.4%.

The results of the challenge of Invaplex_(AR) mice with a lethal dose ofS. flexneri 2a indicate that Invaplex_(AR) stimulates a level ofprotection that is comparable to or exceeds that of native Invaplex.Furthermore, it appears that the complex of IpaB, IpaC and LPS isrequired in that individual components are incapable of stimulating afully effective protective immune response.

TABLE 1 Lethal Challenge of mice immunized with S. flexneri 2aInvaplexAR or native Invaplex¹. Day of Maximum 50% Total Immunizing WgtWgt No. No. No. of P Antigen Loss (%) recovery Survivors Dead Animals %Protection† Value* S. flexneri 2a 21.32 7 15 0 15 100% <0.001Invaplex_(AR) , 2.5 μg Invaplex 24 JWJX, 26.02 13 15 0 15 100% <0.001 5μg Invaplex 24 JWJX, 23.81 7 12 0 12 100% <0.001 10 μg S. flexneri 2aLPS, 29.95 8 3 3 6 40.9%  0.262 2.5 μg Purified 1paB, 30.97 >14 5 1 680.3%  0.010 2.5 μg Purified 33.91 >14 1 5 6  1.5% 1.000 HisTagIpaC, 2.5μg 0.9% Saline 31.35 10 2 11 13  0% — ¹Three weeks after the finalimmunization, mice were intranasally challenged with 1.5 × 10⁷ cfu of S.flexneri 2a,. Weight loss and symptoms were monitored daily for 14 days.†% Protection = [(%Death_(Control) - % Death_(Vaccinees))/%Death_(Control)] × 100 *Fisher Exact Test

Antigen-Specific Mucosal Antibody Response After Intranasal Immunizationwith Invaplex_(AR)

Intestinal and lung washes collected on day 35 from mice immunized withS. flexneri 2a Invaplex_(AR) or native Invaplex were assessed by ELISAfor antigen-specific IgA levels. Immunization with Invaplex_(AR) inducedlevels of LPS and Invaplex-specific intestinal IgA that were comparableto levels induced by immunization with native Invaplex (FIG. 7) andsignificantly higher levels (p<0.001) of IpaB-specific IgA. MinimalIpaC-specific intestinal IgA was elicited after immunization with any ofthe Invaplex vaccine preparations (Tables 7 and 8).

Intranasal immunization with Invaplex_(AR) elicited strong antibodyresponses in the lung, directed to LPS, Invaplex 50, IpaB and IpaC (FIG.8 and Tables 7 and 8). Minimal levels of LPS-specific IgA were inducedin the lung after immunization with native Invaplex with undetectablelevels of antibodies specific for Invaplex 50, IpaB and IpaC.

Antigen-Specific Cellular Proliferative Response and Secreted CytokineProfiles after Intranasal Immunization with Invaplex_(AR)

Splenocytes collected from immunized mice on day 35 were stimulated invitro with Invaplex 24, IpaB, or IpaC to assess induction ofantigen-specific cell-mediated responses. Proliferation of cells afterincubation with antigen indicates prior exposure and immunologicalmemory. Concavalin A (ConA), which non-specifically activates T cellproliferation, was used as a positive control to demonstrate viable celllevels. Stimulation indices (Sis) after stimulation with ConA rangedfrom 13.8 to 15.9 (Table 2). Cells from saline inoculated animals didnot proliferate after incubation with Invaplex, IpaB, or IpaC.Splenocytes from animals (4/4) immunized with Invaplex_(AR) proliferatedafter incubation with Invaplex (SI=10.2), IpaB (SI=8.7), and IpaC(SI=6.9) indicating immunological memory to these antigens was present.The IpaB and IpaC-specific proliferative responses in groups immunizedwith Invaplex_(AR) were significantly higher (P<0.01) than theproliferative responses in groups immunized with native Invaplex.Splenocytes from 4/4 animals immunized with native Invaplex proliferatedafter incubation with Invaplex (SI=6.8). Low to undetectable levels ofproliferation occurred in splenocytes from mice immunized with nativeInvaplex after ex vivo incubation with IpaB (1/4 mice) or IpaC (0/4mice).

TABLE 2 Antigen-specific cellular proliferation of splenocytes from miceintranasally immunized with S. flexneri 2a Invaplex_(AR) or nativeInvaplex after in vitro stimulation. Cellular proliferative responsesafter in vitro stimulation^(a) with: S. flexneri 2a Invaplex 24 IpaBIpaC ConA Grp. Treatment (1 μg/ml) (5 μg/ml) (5 μg/ml) (5 μg/ml) 31 S.flexneri 2a 10.2 ± 3.4^(c)*, ** 8.7 ± 2.2*, ** 6.9 ± 3.9*, ** 14.2 ±4.5*, ** Invaplex_(AR) (4/4) (4/4) (4/4) (4/4) (KR-C5; 2.5 μg)^(b) 32 S.flexneri 2a  6.8 ± 1.5* 2.2 ± 1.4 1.6 ± 0.9 15.9 ± 2.3 native Invaplex(4/4) (1/4) (0/4) (4/4) 24 (JWJX; 2.5 μg) 33 0.9% saline  1.1 ± 0.4 1.2± 0.8 1.8 ± 1.1 13.8 ± 3.3 (0/4) (0/4) (0/4) (4/4) ^(a)proliferativeresponses in splenocytes were determined after stimulation in vitro for3 days with ConA and 5 days with S. flexneri 2a Invaplex 24 (lot JWJX),IpaB or IpaC. ^(b)(lot number; immunization dose) ^(c)Mean stimulationindex ± ISD (number of responders/total number in group) *P < 0.05 ascompared to saline-inoculated group (unpaired t test). **P < 0.01 ascompared to native Invaplex-immunized group (unpaired t test).

Confirmation of Protection of Mice from a Lethal Challenge of S.flexneri after Intranasal Immunization with Invaplex_(AR) using aDifferent Lot of Invaplex_(AR)

The experiment describing protection with Invaplex_(AR) in the mouselethal lung model was repeated with a different lot of S. flexneri 2aInvaplex_(AR). In addition the evaluation of the purified components wasrepeated along with mice immunized with mixtures of two purifiedcomponents (IpaB+IpaC; IpaB+LPS; IpaC+LPS). For this challenge, micewere inoculated intranasally with a slightly higher challenge dose(1.6×10⁶ cfu) of S. flexneri 2a (strain 23457T). In naive mice (treatedwith saline) 14 of 14 mice died with a mean maximum weight loss of 34.5%of the pre-challenge weight (see Table 3). Mice immunized with S.flexneri 2a Invaplex_(AR) (13 of 14 survived; p<0.001) survived thelethal challenge with S. flexneri whereas mice immunized with nativeInvaplex had a much lower survival rate for the challenge dose used inthis experiment (See Table 3). With respect to weight loss miceimmunized with Invaplex_(AR) lost less of their pre-challenge weight(26.6%) as compared to 31.6% weight loss in the nativeInvaplex-immunized mice and 34.5% in the mice inoculated with saline.

The protective capacity of the individual components (IpaB, IpaC andLPS) or pairs of purified components used to construct Invaplex_(AR)confirmed previous results in that IpaC and LPS are not protective.Purified IpaB was not protective either. (see Table 3). Mixtures of twoof the purified components resulted in protection in the IpaB+LPScombination and the IpaC+LPS combination whereas the IpaB+IpaCcombination was not fully protective.

The results of the second experiment evaluating the protective capacityof Invaplex_(AR) clearly shows that Invaplex_(AR) stimulates a level ofprotection that exceeds that of native Invaplex. Furthermore, it appearsthat the individual components (IpaB, IpaC or LPS) are incapable ofstimulating a fully effective protective immune response.

TABLE 3 Lethal Challenge of mica immunized with S. flexneri 2aInvaplexAR or native Invaplex¹ Maximum Day of 50% Wgt Wgt No. Total No.of Antigen Loss (%) Recovery Survivors No. Dead¹ Animals % Protection² PValue³ IVP_(AR) S.flex 2a 26.6 10  13 1 14 92.9 <0.001 KR-C5, 2.5 μgIVP₂₄ S.flex2a 31.6 — 4 10 14 28.6 0.098 JWJX,5 μg 0.9% Saline 34.5 — 014 14 0 S.flex2a LPS, 32.7 — 0 14 14 0 NS 2.5 μg IpaB, 2.5 μg 29.8 13  113 14 7.1 NS HisTag IpaC, 38 — 0 14 14 0 NS 2.5 μg S.flex2a LPS/ 24 8 140 14 100 <0.001 IpaB, 2.5 μg each S.flex2a LPS/ 25 8 13 1 14 92.9 <0.001HisTag IpaC, 2.5 μg each IpaB/HisTag 26.7 8 5 9 14 37.7 0.041 IpaC, 2.5μg each ¹Three weeks after the final immunization, mice wereintranasally challenged with 1.6 × 10⁷ cfu of S. flexneri 2a,. Weightloss and symptoms were monitored daily for 14 days. ²% Protection = [(%Death_(Control) - % Death_(Vaccinees))/% Death_(Control] × 100) ³FisherExact Test

S. sonnei Invaplex_(AR) Murine Immunogenicity and Protection Study

Serum antibody responses directed to S. sonnei Invaplex 50, S. sonneiLPS, IpaB and IpaC were determined by ELISA. Mice inoculated with saline(negative control) did not have detectable levels of antigen-specificserum IgG or IgA on day 42 (Table 4). Similar S. sonnei Invaplex 50 andLPS-specific serum IgG and IgA endpoint titers were achieved afterimmunization with S. sonnei Invaplex_(AR) and native Invaplex (Table 4).Groups of mice immunized with S. sonnei Invaplex_(AR) had higher levelsof IpaB-specific serum IgG (GMT>5760, P<0.001) as compared to groups ofmice immunized with native S. sonnei Invaplex (GMT 546). Animalsimmunized with Invaplex_(AR) had an anti-IpaC serum IgG mean titer of1091 (P<0.001) whereas animals immunized with native Invaplex or salinehad undetectable levels of IpaC-specific IgG.

TABLE 4 Antigen-specific serum IgG and IgA endpoint titers on day 42 inmice after intranasal immunization with S. sonnei Invaplex_(AR) ornative S. sonnei Invaplex 50 S.sonnei Invaplex 50 S. sonnei LPS IpaBIpaC IgG IgA IgG IgA IgG IgG Saline 90 ± 0^(c,d) 45 ± 0  90 ± 0 45 ± 090 ± 0  90 ± 0 Native 2183 ± 789   1254 ± 322  103 ± 40 157 ± 40 546 ±483 90 ± 0 S. sonnei Invaplex 50 (5 μg)^(a) Artificial 5014 ± 1288**2864 ± 36**  136 ± 117  313 ± 224* >5760**  1091 ± 1172** S. sonneiInvaplex_(AR) (2.5 μg)^(b) ^(a)Group 6 ^(b)Group 3 ^(c)Geometric meanendpoint titer ± 1 standard deviation ^(d)Comparisons between groupswere accomplished using two-way ANOVA analysis of naturallog-transformed endpoint titers. *p < 0.05 as compared with native S.sonnei Invaplex 50 **p < 0.001 as compared with native S. sonneiInvaplex 50

Protection of Mice from a Lethal Challenge of S. sonnei or HeterologousS. flexneri after Intranasal Immunization with S. sonnei Invaplex_(AR)

The protective capacity of InvaplexAR was also evaluated for anInvaplexAR manufactured from purified S. sonnei LPS and recombinant IpaBand IpaC using the mouse lethal lung model. It was compared to native S.sonnei Invaplex. In addition the capacity of S. sonnei Invaplex_(AR) toprotect against a heterologous S. flexneri challenge was also evaluated.In nave mice (treated with saline) 15 of 15 mice died with a meanmaximum weight loss of 23.4% of the pre-challenge weight (see Table 5).Mice immunized with S. sonnei Invaplex_(AR) survived (15 of 15 survived;p<0.001) the lethal challenge with S. sonnei whereas mice immunized withnative Invaplex also exhibited solid protection (14 of 14 survived,P<0.001) (See Table 5). With respect to weight loss, mice immunized withS. sonnei Invaplex_(AR) lost 7.7% of their pre-challenge weight ascompared to 9.2% weight loss in the native S. sonnei Invaplex-immunizedmice and 23.4% in the mice inoculated with saline.

Interestingly, the S. sonnei Invaplex_(AR) also provided significantprotection (15 or 15 challenged mice survived, P<0.001)) against aheterologous S. flexneri 2a challenge suggesting that the immuneresponse to either IpaB or IpaC may contribute significantly to theprotective immune response.

The results of this experiment evaluating the protective capacity of S.sonnei Invaplex_(AR) clearly shows that Invaplex_(AR) from anotherShigella species stimulates a level of protection that is comparable tothat of native Invaplex.

TABLE 5 Lethal Challenge of mica immunized with S. sonnei Invaplex_(AR)or native S. sonnei Invaplex¹. Stimulation of homologous andheterologous immunity Day of Maximum 50% Challenge Wgt Loss Wgt No. No.Total No. of % P Antigen Agent (%) Recovery Survivors Dead¹ AnimalsProtection² Value³ Saline S. sonnei 23.4 — 0 15 15 0 — Sonnei IVP- S.sonnei 7.7 3 15 0 15 100 <0.001 AR-KJD3D6 2.5 ug IVP50 JOJP S. sonnei9.2 7 14 0 14 100 <0.001 5 ug 3x Saline S. flexneri 35.1 — 0 15 15 — —2a Sonnei IVP- S. flexneri 31.0 9 15 0 15 100 <0.001 AR 2.5 ug 2a S.flex 2a S. flexneri 25.0 — 11 3 14 78.6 <0.001 JWJX 5 ug 3x 2a (Nasal)¹Three weeks after the final immunization, mice were intranasallychallenged with 1.6 × 10⁷ cfu of S. flexneri 2a or 8 × 10⁶ cfu S.sonnei, as indicated. Weight loss and symptoms were monitored daily for14 days. ²% Protection = [(% Death_(Control) - % Death_(Vaccinees))/%Death_(Control)] × 100 ³Fisher Exact Test

Protection of Guinea Pigs with S. flexneri 2a Invaplex_(AR) using theGuinea Pig Keratoconjunctivitis Model

The guinea pig keratoconjunctivitis model is used to evaluate Shigellavaccines and is often used as the precursor to human trials. The modelinvolves infection of the guinea pig eyes with Shigella which establishinfection in the corneal epithelium. This outcome (severekeratoconjunctivitis) is a result of invasion of the corneal epitheiliumby the shigellae and the subsequent inflammatory response by the hostnot unlike that observed in the human intestinal tract.

In this experiment guinea pigs were immunized three time intranasallywith either S. flexneri 2a InvaplexAR or native Invaplex. A salinecontrol group was also utilized. Three weeks after the finalimmunization animals were challenged ocularly with S. flexneri 2a. Boththe InvaplexAR (90% protection, P<0.001) and native Invaplex (100%protection, P<0.001) provided solid protection (See Table 6) indicatingthat the Invaplex_(AR) is comparable to the native Invaplex.

TABLE 6 Protection against Shigella infection in guinea pigs with S.flexneri 2a Invaplex_(AR) using the keratoconjunctivitis model. %Treatment # positive¹ # Negative protection² P value³ S. flexneri 2anative 0 10 100%  <0.001 Invaplex 50, 25 μg/dose S. flexneri 2a 1 9 90%<0.001 Invaplex_(AR), 25 μg/dose IpaB (25 μg) + IpaC 4 6 40% 0.01 (25μg) per dose IpaB (25 μg) + IpaC 0 10 100%  <0.001 (25 μg) + LPS (25 μg)per dose (0.9% Saline) 10 0  0% — ¹Guinea pigs were challengedintraocularily with 1 × 10⁸ cfu of S. flexneri 2a. Eyes were evaluatedon day 5 post-infection for keratoconjunctivitis as described by Hartmanet al. [44] ²% Protection = [(%Disease_(Control) − %Disease_(Vaccinees))/% Disease_(Control)] × 100 ³Fisher Exact Test

S. flexneri 2a Invaplex_(AR) Murine Adjuvanticity Study

Intranasal immunization with OVA alone or OVA combined with CT orpre-immune samples from immunized animals (day 0) did not inducedetectable levels of serum IgG or IgA with specificity to S. flexneri 2aInvaplex 50, S. flexneri 2a Invaplex 24 (FIG. 9), S. flexneri 2a LPS(FIG. 10), IpaB or IpaC (FIG. 11). Immunization with OVA combined witheither Invaplex_(AR) or native Invaplex induced similar Invaplex 50,Invaplex 24 and LPS-specific serum IgG and IgA endpoint titers (FIGS. 9and 10. Endpoint titers against purified IpaB (p<0.05) and IpaC(p≦0.001) were higher in mice immunized with OVA+Invaplex_(AR) ascompared to mice immunized with OVA+native Invaplex (FIG. 11).Comparable levels of OVA-specific serum IgG and IgA were induced in miceimmunized with OVA combined with Invaplex_(AR), native Invaplex, or CTon day 42 and were significantly higher (p≦0.001) than the responsesinduced in mice after immunization with OVA alone (FIG. 12).

Antigen-specific antibodies in lung washes were also assessed by ELISAto investigate the mucosal immune responses induced after immunization(FIG. 13). Immunization with OVA combined with Invaplex_(AR) inducedsimilar levels of LPS and Invaplex 24-specific IgG and IgA in lungwashes as compared to responses after immunization with OVA combinedwith native Invaplex and higher than the levels induced afterimmunization with OVA alone, or OVA combined with CT. Similar levels ofOVA-specific lung IgG and IgA were induced after immunization with OVAcombined with Invaplex_(AR), native Invaplex, or CT which weresignificantly (p<0.001) higher than those induced after immunizationwith OVA alone. Moderate levels of LPS and Invaplex-specific IgA inintestinal washes (data not shown) were detected in washes from miceimmunized with OVA combined with Invaplex_(AR) or native Invaplex andwere undetectable in mice immunized with OVA alone or OVA combined withCT. OVA-specific intestinal IgA responses were below levels of detectionin all samples assayed.

TABLE 7 Anti-IpaB and anti-IpaC serum (day 42) and mucosal (day 35)antibody levels after intranasal immunization of mice with S. flexneri2a Invaplex or Invaplex_(AR) Anti-IpaB Anti-IpaC Study Serum LungIntestinal Serum Lung Intestinal Name Treatment IgG^(a) IgA IgA IgG IgAIgA S. flexneri 2a Invaplex >5760 ND ND 136 ± 117 ND ND Invaplex_(AR) (5μg) (>5760) (90–360) Mouse Study I Invaplex_(AR) >5760 ND ND 1440 ±4593* ND ND (2.5 μg) (>5760) ND ND (90–11520) ND ND Saline 90 ± 0 90 ±90) (90 − 90 (90 − 90 S. flexneri 2a Invaplex TBD 2 ± 0 3 ± 1 TBD 2 ± 02 ± 0 Invaplex_(AR) (5 μg) (2 − 2) (2 − 4) (2 − 2) (2 − 2) Mouse StudyII Invaplex_(AR) TBD 54 ± 58* 64 ± 54* TBD 5 ± 15 2 ± 0 (2.5 μg) (8 −128) (16 − 128) (2 − 32) (2 − 2) Saline TBD 2 ± 0 2 ± 0 TBD 2 ± 0 2 ± 0(2 − 2) (2 − 2) (2 − 2) (2 − 2) S. flexneri 2a Invaplex + 2507 ± 2366 NDND 157 ± 148 ND ND Invaplex_(AR) OVA (90 − 90) (90 − 360) AdjuvanticityInvalpex_(AR) + >5760* ND ND 950 ± 2318* ND ND Study OVA (>5760) (90 ±5760) OVA 90 ± 0 ND ND 90 ± 0 ND ND (90 − 90) (90 − 90) ^(a)Serumresponses in the S. flexneri 2a Invaplex_(AR) Mouse Study I are bllodcollected on day 42. Serum responses in the S. flexneri 2a Invaplex_(AR)Adjuvanticity Study are from blood collected on day 35. ^(b)Geomean ± 1standard deviation from the mean (n = 5 mice/group) (range of endpointtiters) *Significantly higher endpoint titers (one way ANOVA of logtransformed endpoint titers; p ≦ 0.001) as compared to endpoint titersof animals immunized with native Invaplex. ND; not determined, TBD; tobe determined

TABLE 8 Antigen-specific serum IgG and IgA endpoint titers on day 42 inguinea pigs intranasally immunized with native Invaplex, Invaplex_(AR),or mixtures of IpaB and IpaC. ELISA Antigen S. flexneri 2a Invaplex 50S. flexneri 2a LPS IpaB IpaC Treatment IgG IgA IgG IgA IgG IgG S.flexneri 2a 827 ± 1046^(b) 475 ± 1148 1091 ± 2873 827 ± 1018 1712 ± 230590 ± 0 Invaplex (180 − 2880) (180 − 2880) (180 − 5760) (360 − 2880) (720−≧ 5760) (90 − 90) (lot 1307; 25 μg) S. flexneri 2a 4073 ± 2160* 856 ±360 2880 ± 2700 827 ± 1046 All > 5760* All > 5760* Invaplex_(AR) (1440−≧ 5760) (720 − 2880) (360 −≧ 5760) (720 − 2880) (>5760) (>5760) (lotKR-C5; 25 μg) IpaB + IpaC 1211 ± 1034 255 ± 104 90 ± 0 45 ± 0 All >5760*3629 ± 2494* (25 μg + 25 μg) (360 − 2880) (180 − 360) (90 − 90) (45 −45) (>5760) (1440 −≧ 5760) Saline 90 ± 0 45 ± 0 90 ± 0 45 ± 0 90 ± 0 90± 0 (90 − 90) (45 − 45) (90 − 90) (45 − 45) (90 − 90) (90 − 90)^(a)Serum responses in the S. flexneri 2a GMP Stability andInvaplex_(AR) guinea pig study are from blood collected on day 42.

Method for Formation and Evaluation of Artificial Invaplex Complexedwith an Antibiotic or other Therapeutic Molecule for IntracellularDelivery

Many therapeutic biochemicals, including antibiotics, are oftenineffective against intracellular target because they are unable tocross mammalian cell membranes or because they require highextracellular concentrations to achieve therapeutic concentrationsinside of mammalian cells. The artificial Invaplex provides a mechanismto create a complex of invasins, LPS and antibiotic by mixing thecomponents during the assembly stage for creating artificial Invaplex.Once assembled, the native properties of Invaplex allow it to transportthe complexed antibiotic or therapeutic molecule into mammalian cells.

Purified, soluble IpaB and IpaC, each in their respective final buffersare mixed together at an IpaC/IpaB molar ratio of 8. After the IpaB andIpaC are mixed, a solution of antibiotic, for example gentamicin at 5 to100 μg/ml, is added to the mixture. Next the IpaB, IpaC and antibioticsolution is slowly added to dry LPS powder (ratio of LPS to totalprotein is 0.56). LPS from any Shigella species can be used; for thedescribed experiments S. flexneri 2a, S. sonnei or S. dysenteriae 1 LPSwas used. The mixture is incubated at 37° C. for approximately 2 hourswith shaking. Afterwards the protein/LPS/antibiotic mixture is dilutedto 20 mM Tris-HCl, 0.10 M NaCl and 1.2 M urea to reduce the NaClconcentration prior to ion-exchange chromatography. For finalpurification, the diluted IpaB/IpaC/LPS/antibiotic mixture is applied toan anion exchange column (HiTrap™ Q HP) with Buffer A consisting of 20mM Tris-HCl, pH 9.0 and Buffer B consisting of 1 M NaCl, 20 mM Tris-HCl,pH 9.0, and a step gradient of 0% (5 column volumes) to 24% (10 columnvolumes) to 50% (6 column volumes) to 100% Buffer B (5 column volumes).One ml fractions were collected from a 1 ml HiTrap™ Q HP column at anelution flow rate of 1 ml/min. Fractions from each step were analyzedfor the presence of IpaB, IpaC and LPS by spotblot. The fractionscontaining IpaB, IpaC and LPS are the artificial Invaplex fractions.

The effectiveness of the artificial Invaplex-antibiotic complex will beevaluated in its ability to kill intracellular microorganisms such asshigellae growing in tissue culture cells. The artificialInvaplex-antibiotic complex will be incubated with Shigella-infectedtissue culture cells. After 2 to 24 hours the number of intracellularShigella will be determined by plating lysates of the treated cells (anduntreated control cells) on trypticase soy agar plates to determine thelevel intracellular killing in the cells treated with the artificialInvaplex-antibiotic complex.

Further variations and modification of the foregoing will be apparent tothose skilled in the art and are intended to be encompassed by theclaims appended thereto.

REFERENCES

The references which follow are identified by number within thespecification. The contents of each of these documents are expresslyincorporated herein to the degree necessary to understand the invention.

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1. An artificial Invaplex consisting of IpaB:IpaC complex complexed witha purified lipopolysaccharide (LPS) from a gram negative bacteriaselected from S. flexneri, S. sonnei, S. dysenteriae, S. boydii,enteroinvasive E. coli, Yersinia or Salmonella, wherein the artificialInvaplex is prepared by mixing IpaB and IpaC to form a complex,IpaB:IpaC, and then mixing IpaB:IpaC with the lipopolysaccharide to formthe artificial Invaplex and wherein the IpaB and the IpaC arerecombinantly produced and of Shigella origin.
 2. The artificialInvaplex of claim 1, wherein the lipopolysaccharide is from a gramnegative bacteria selected from S. flexneri, S. sonnei, S. dysenteriae,S. boydii, or enteroinvasive E. coli.
 3. The artificial Invaplex ofclaim 2, wherein the lipopolysaccharide is from a gram negative bacteriaselected from S. flexneri, S. sonnei, S. boydii, or S. dysenteriae. 4.The artificial Invaplex of claim 1, wherein the LPS is from a Shigellaserotype.
 5. The artificial Invaplex of claim 1, wherein the IpaB ispurified recombinant IpaB.
 6. The artificial Invaplex of claim 1,wherein the IpaC is purified recombinant IpaC.
 7. The artificialInvaplex of claim 1, wherein the IpaC, IpaB and lipopolysaccharide arepresent in amounts that correspond to those of native Invaplex
 24. 8.The artificial Invaplex of claim 1, wherein the lipopolysaccharideincludes two or more lipopolysaccharides selected from at least twodifferent serotypes of gram negative bacteria.
 9. The artificialInvaplex of claim 1, wherein the IpaC and IpaB are present in anIpaC:IpaB ratio in the range of 0.08:1 to 80:1.
 10. The artificialInvaplex of claim 9, wherein the IpaC:IpaB ratio is 0.8:1 to 20:1. 11.The artificial Invaplex of claim 10, wherein the IpaC:IpaB ratio isapproximately 8:1.
 12. The artificial Invaplex of claim 1, wherein theLPS is present relative to the total protein (IpaB and IpaC) in anLPS:protein ratio selected from the range of 0.01:1 to 10:1.
 13. Theartificial Invaplex of claim 12, wherein the LPS:protein ratio range isfrom 0.5:1 to 5:1.
 14. The artificial Invaplex of claim 13, wherein theLPS:protein ratio is approximately 0.5:1.
 15. The artificial Invaplex ofclaim 1, further comprising a labeled detectant, antibiotic, therapeuticor biomolecule.
 16. The artificial Invaplex of claim 15, wherein thebiomolecule includes proteins, enzymes, polysaccharide, or nucleicacids.
 17. A composition comprising the artificial Invaplex of claim 1and a pharmaceutically acceptable carrier.
 18. The composition of claim17 further comprising an immunogen.
 19. The composition of claim 17,wherein the artificial Invaplex present is sufficient to enhance animmune response to an immunogen.
 20. The composition of claim 17,wherein the amount of artificial Invaplex present is sufficient toinduce a protective immune response.
 21. The composition of claim 17,wherein the artificial Invaplex further comprises a labeled detectant,antibiotic, therapeutic or biomolecule.
 22. The composition of claim 21,wherein the biomolecule includes proteins, enzymes, polysaccharide ornucleic acids.
 23. The composition comprising the artificial Invaplex ofclaim 8 and a pharmaceutically acceptable carrier.